The American Cancer Society estimates that more than 40,000 new cases of invasive cancer in the oral cavity and pharynx are reported each year in the US, and five-year survival rates remain below 40% for advanced stage disease. The goal of this research is to design, build and test a structured illumination-enabled multispectral widefield fluorescence lifetime imaging microendoscope for detection of oral precancer in vivo. Combining structured illumination (SI) with fluorescence lifetime imaging (FLIM) would enable generation of high fidelity three dimensional volumes of the fluorescence intensity and lifetime of the epithelial tissue. We believe that the ability to collect 'optical sections' across the epithelum would allow characterization of tissue biochemical composition due to differential distribution of molecular markers, such as structural proteins and metabolic cofactors, within tissue and will ultimately aid in high sensitivity and specificity detection of early oral cancer development. The specific aims of this proposal are to: 1) construct a structured illumination multispectral FLIM endoscope (SI-FLIM) system to provide optical depth sectioning; 2) characterize the performance and optimize the design specifications of the SI-FLIM endoscope by imaging in vitro and ex vivo; and 3) validate and demonstrate the capability of SI-FLIM by imaging tissue in vivo. The successful completion of this work will enable the development of the first multispectral SI-FLIM microendoscope with the ability to selectively image specific depths within the tissue, and will demonstrate its potential for non-invasive characterization of oral premalignant and malignant lesions. With the wealth of specific molecular information encoded in the collected fluorescence intensity and lifetime in multiple spectral channels, such a device can serve to provide an objective screening of the oral cavity in addition to the clinician's subjective evaluation. Although the proposed endoscope is designed for application of this device in the oral cavity, the SI-FLIM system can be applicable to detect and diagnose other carcinomas (e.g. basal cell, colorectal, and cervical) as well with slight modifications to the endoscope.